Celiprolol Reduces Oxidative Stress and Attenuates Left Ventricular Remodeling Induced by Hypoxic Stress in Mice

ORIGINAL ARTICLE

Celiprolol reduces oxidative stress and attenuates left ventricular remodeling induced by hypoxic stress in mice

Satoshi Nishioka1, Toshitaka Yoshioka1, Atsuo Nomura2, Ryuji Kato2, Masatoshi Miyamura3, Yoshikatsu Okada4, Nobukazu Ishizaka3, Yasuo Matsumura1 and Tetsuya Hayashi2

We have previously reported that intermittent hypoxic stress, which is relevant to sleep apnea syndrome (SAS), increases oxidative stress and induces left ventricular (LV) remodeling. Celiprolol, a b1-selective adrenoreceptor blocker, is known to have not only an antihypertensive effect but also an antioxidant effect through releasing nitric oxide. The aim of this study was to examine the hypothesis that celiprolol might ameliorate the LV remodeling induced by intermittent hypoxia through its antioxidant effect. Male C57BL/6J mice (8 weeks old) were exposed to intermittent hypoxia (30 s of 5% oxygen followed by 30 s of 21% oxygen) for 8 h day À1 during the daytime for 10 consecutive days or were maintained under normoxic conditions. Animals were treated with either celiprolol (100 mg kg À1 day À1 by gavage) or vehicle. Hypoxic stress caused fluctuations in blood pressure (BP), an increase in the mean cardiomyocyte diameter, perivascular fibrosis and a decrease in endothelial nitric oxide synthase (eNOS) expression. These changes were associated with increased levels of 4-hydroxy-2-nonenal protein, superoxide, tumor necrosis factor-a mRNA and brain natriuretic peptide mRNA in the LV myocardium. Celiprolol significantly suppressed BP fluctuation, restored eNOS expression and reduced oxidative stress and superoxide production, thus ameliorating hypoxia-induced LV remodeling in mice. These findings suggest that treatment with celiprolol might prevent cardiovascular events in borderline hypertensive patients with SAS. Hypertension Research (2013) 36, 934–939; doi:10.1038/hr.2013.60; published online 20 June 2013

Keywords: celiprolol; intermittent hypoxia; oxidative stress

INTRODUCTION patients with moderate-to-severe SAS.10 In addition, both blood Sleep apnea syndrome (SAS) is a risk factor for cardiovascular disease, pressure (BP) and sympathetic nerve activity are increased during being associated with hypertension, coronary artery disease and sleep in SAS patients.11 1–4 stroke. Major pathophysiological features of SAS include the Some b1-selective adrenoreceptor blockers are known to have not production of reactive oxygen species (ROS) and inflammatory only an antihypertensive effect but also an antioxidant effect. 5,6 cytokines induced by intermittent hypoxia. It has been reported Celiprolol is a b1-selective adrenoreceptor blocker with intrinsic that ROS can cause endothelial dysfunction, cardiomyocyte sympathomimetic activity that also promotes the release of nitric hypertrophy and interstitial fibrosis.7 We have previously oxide (NO) and decreases ROS production.12–14 However, the effect demonstrated that gp91phox-containing NADPH (nicotinamide of celiprolol on LV remodeling induced by intermittent hypoxia adenine dinucleotide phosphate reduced) oxidase has a crucial role remains unclear. Thus, the aim of this study was to examine the in the development of left ventricular (LV) remodeling induced by hypothesis that celiprolol might ameliorate LV remodeling induced intermittent hypoxia in mice.8 In addition, hypoxic stress enhances by intermittent hypoxia through its antioxidant effect. atherosclerosis and LV remodeling in apolipoprotein E-knockout mice, while treatment with an angiotensin II receptor blocker METHODS reduces oxidative stress and ameliorates interstitial fibrosis in the Experimental protocol 9 LV myocardium. In clinical studies, diastolic dysfunction and a Eight-week-old male C57BL/6J mice (n ¼ 60) were purchased from Clea Japan, decrease in the myocardial contractile reserve, suggesting myocardial Inc. (Osaka, Japan) for this study. The mice were exposed to a 12-h light–dark degeneration and/or interstitial fibrosis, have been observed in cycle, given free access to tap water and fed standard chow ad libitum.

1Laboratory of Pathological and Molecular Pharmacology, Osaka University of Pharmaceutical Sciences, Osaka, Japan; 2Laboratory of Cardiovascular Pharmacotherapy and Toxicology, Osaka University of Pharmaceutical Sciences, Osaka, Japan; 3Department of Internal Medicine III, Osaka Medical College, Osaka, Japan and 4Department of Pathology, Osaka Medical College, Osaka, Japan Correspondence: Dr T Hayashi, Laboratory of Cardiovascular Pharmacotherapy and Toxicology, Osaka University of Pharmaceutical Sciences, Nasahara 4-20-1, Takatsuki, Osaka 569-1094, Japan. E-mail: [email protected] Received 6 December 2012; revised 25 March 2013; accepted 11 April 2013; published online 20 June 2013 Celiprolol and hypoxic stress SNishiokaet al 935

The hypoxic stress group was housed in a chamber and exposed to intermittent necrosis factor-a (TNF-a)) purchased from Applied Biosystems. The reaction hypoxia (30 s of 4.5–5.5% oxygen followed by 30 s of 21% oxygen) for conditions were 50 1C for 2 min and 95 1C for 10 min, followed by 40 cycles 8 h day À1 during the daytime for 10 consecutive days (IH mice). Control of 95 1C for 15 s and 60 1C for 1 min. The amount of target mRNA was animals were housed in identical cages in the same room and were exposed to normalized for that of 18 s mRNA.17 the same ambient noise and lighting conditions as the IH mice. The animals were administered celiprolol (100 mg kg À1 day À1) or its vehicle by oral gavage. Statistical analysis After the 10-day experimental period, cardiac catheterization was performed Results are expressed as the mean±s.e.m. Variables were compared between under anesthesia with intraperitoneal pentobarbital sodium (50 mg kg À1)to groups by one-way analysis of variance followed by the Tukey–Kramer multiple measure the biventricular systolic pressure.9 Then the heart was excised, and comparison test, and Po0.05 was considered to indicate significance. the upper half was used for light microscopy, while the LV free wall was excised for immunohistochemistry and the reverse transcription PCR. The experimental protocol and handling of animals during experiments were RESULTS approved by the Experimental Animal Research Committee of Osaka Body weight, heart weight and hemodynamics University of Pharmaceutical Sciences. There were no significant differences in body weight, heart weight, HR and biventricular systolic and LV end-diastolic pressures among Monitoring of BP and heart rate (HR) the groups (Table 1). The arterial pressure was monitored during hypoxic stress by using a Data-quest IV radiotelemetry system (Data Sciences International, St Paul, MN, USA). Under anesthesia, a thoracic aortic catheter was implanted in Changes in BP during intermittent hypoxia each mouse and was attached to a PA-C10 radiotelemetry transmitter. During the hypoxic interval, there was an instantaneous surge in BP The transmitter was secured in a subcutaneous pocket for the duration of associated with an increased HR, and BP returned to baseline after a the experiment. After 1 week for recovery from surgery, the BP and HR were few seconds. Reoxygenation with room air caused a similar BP surge recorded throughout the experiment. and tachycardia for several seconds, after which the BP and HR returned to the basal levels. These BP fluctuations, associated with an Histological examination increased s.d. of the mean BP, were observed during intermittent Isolated ventricular tissues were fixed in 10% formaldehyde, embedded in hypoxia throughout the experiments (Figure 1). The mean BP showed paraffin and cut into 4-mm sections. To evaluate the mean cardiomyocyte elevation from 3 days after the initiation of hypoxic stress and was diameter, the shortest diameter of each nucleated cardiomyocyte was measured significantly elevated on the tenth experimental day. Treatment with on transverse sections stained with hematoxylin–eosin under a light micro- celiprolol suppressed these BP and HR fluctuations, although the 10 scope at a Â 400 magnification. After staining with Sirius Red, color images mean BP was elevated during hypoxic stress (Figure 2). of each section were taken with a digital camera (Fujix Digital Camera HC-300Z; Fujifilm, Tokyo, Japan) mounted on a Nikon Microphot-FXA (Nikon, Tokyo, Japan), and the percentage of the area of interstitial fibrosis was Histological findings calculated by the previously described method.9 Exposure to intermittent hypoxia led to an increase in the mean cardiomyocyte diameter, myofiber disarray and interstitial fibrosis Immunohistochemistry for 4-hydroxy-2-nonenal (4-HNE), in the LV myocardium. In particular, the percentage of the area of macrophages, endothelial nitric oxide synthase (eNOS) and perivascular fibrosis was significantly increased (Figure 3). A small arginase II number of CD68 ( þ ) macrophages were observed in the vicinity of Immunohistochemical staining was performed as described previously9 Briefly, arteries. sections cut from paraffin blocks were incubated with antibodies targeting Treatment with celiprolol prevented these histological changes 4-HNE (No. MHN-20; Japan Institute for the Control of Aging, Shizuoka, caused by hypoxic stress. Japan), macrophages (anti-CD68, No.ab125212; Abcam, Tokyo, Japan), arginase II (H-64, Santa Cruz Biotechnology, Dallas, TX, USA) or eNOS Superoxide and 4-HNE (BD Biosciences, San Jose, CA, USA). Then the percentage of the area of 4-HNE, eNOS or arginase II staining was measured by the method described IH mice showed an increase in superoxide production (detected by previously.10 DHE labeling) and an increase in 4-HNE-modified protein adducts in the LV myocardium. Treatment with celiprolol significantly Detection of superoxide in the LV myocardium To evaluate in situ superoxide production, fresh frozen sections of LV Table 1 Effect of hypoxia on body weight, heart weight and myocardium were stained with dihydroethidium (DHE; Molecular Probes, hemodynamics 15 Eugene, OR, USA). Frozen sections (20-mm thick) were covered with 10 mM DHE solution and incubated at 37 1C for 30 min in a humidified chamber with Normoxia Hypoxia protection from light. Then the sections were observed under a fluorescence microscope (Keyence BZ-8000; Osaka, Japan), and the fluorescence intensity of Parameters Vehicle Celiprolol Vehicle Celiprolol DHE was quantified with NIH Image 1.61 software (National Institute of Mental Health, Bethesda, MD, USA) and expressed as a percentage of the total BW, g 22.0±0.8 (8) 23.6±0.5 (8) 22.1±0.5 (13) 22.1±0.4 (13) 16 cross-sectional area. HW, mg 111±3 (8) 112±5(8) 106±3(13) 105±3(13) HW/BW, mg g À1 5.1±0.2 (8) 4.8±0.2 (8) 4.8±0.1 (13) 4.7±0.2 (13) Quantitative real-time reverse transcription PCR HR, b.p.m. 461±28 (8) 492±34 (5) 517±27 (7) 425±29 (10) Total RNA was extracted from myocardial tissue using an RNeasy mini kit RVsys, mm Hg 26±4 (7) 33±4(5) 33±6(8) 27±2(10) (Qiagen, Valencia, CA, USA). Reverse transcription was performed with LVsys, mm Hg 101±2 (7) 102±3(5) 108±3(8) 103±3(8) random hexamers and superscript reverse transcriptase (Invitrogen, Carlsbad, LVed, mm Hg 7±4(7) 5±1(5) 9±4(8) 5±1(7) CA, USA). Real-time quantitative PCR was done with an ABI Step One Abbreviations: BW, body weight; HW, heart weight; HR, heart rate; LVed, left ventricular sequence detector (PE Applied Biosystems, Foster City, CA, USA), using a end-diastolic pressure; LVsys, LV systolic pressure; RVsys, right ventricular systolic pressure. Taqman probe and primers for the target mRNA (Ma00443258 for tumor Values are means±s.e.m. Number of mice in parenthesis.

Hypertension Research Celiprolol and hypoxic stress S Nishioka et al 936

3rd experimental day 10th experimental day

(mmHg) Hypoxia Reoxygenation (mmHg) Hypoxia Reoxygenation 180 180

140 140

Vehicle 100 100

60 60 30 sec 30 sec 30 sec 30 sec

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100 100 Celiprolol

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Figure 1 Monitoring of BP by telemetry. Arterial pressure was monitored in mice during hypoxic stress using a Data quest IV radiotelemetry system (Data Sciences International). BP and HR fluctuations were observed during intermittent hypoxia, while celiprolol suppressed these fluctuations. A full color version of this figure is available at the Hypertension Research journal online.

(mmHg) suppressed the ﬂuctuation of BP and HR during intermittent 130 Vehicle (n=4) ## hypoxia, reduced superoxide production and decreased the Celiprolol (n=3) ** 120 expression of TNF-a and BNP mRNA in the LV myocardium, P=0.1264 consequently attenuating the histological changes caused by chronic 110 # * intermittent hypobaric hypoxia. À1 100 Although treatment with celiprolol at 100 mg day did not affect the hemodynamics at the time of killing, this agent signiﬁcantly 90 reduced oxidative stress and superoxide production. We previously

Mean Arterial Blood Pressure reported that oxidative stress mediated by NADPH oxidase has an 80 important role in LV remodeling caused by intermittent hypoxia.8,17,18 0 Therefore, celiprolol might attenuate the perivascular fibrosis and 03 10 myocardial degeneration observed in mice exposed to hypoxia by Day of Hypoxic Exposure reducing the impact of oxidative stress on the LV myocardium. Figure 2 Mean arterial BP during intermittent hypoxia monitored by In the present study, celiprolol suppressed the intermittent telemetry. The mean BP showed elevation from 3 days after the initiation of hypoxia-induced changes in arginase II and eNOS expression. It is hypoxic stress, and was significantly elevated on the tenth experimental day. L *Po0.05, **Po0.01 vs. Day 0; #Po0.05, ##Po0.01 vs. Day 3. well known that NO is produced from -arginine by NOS, while arginase competes with NOS for L-arginine to produce urea and L-ornithine. Increased arginase expression has been observed in a suppressed superoxide production and the accumulation of 4-HNE number of conditions, including vascular dysfunction,19 pulmonary adducts in the LV myocardium (Figures 4 and 5a). hypertension,20 coronary artery dysfunction21 and hypertension due to chronic hypobaric hypoxia.22 The upregulation of arginase activity TNF-a and brain natriuretic peptide (BNP) might reduce the availability of L-arginine for NOS, leading to a IH mice showed an increase in the expression of TNF-a and BNP decrease in NO production.23 Thus, the downregulation of arginase II mRNA, whereas these changes were significantly suppressed by observed in our study might be important for the vasodilatory action treatment with celiprolol (Figures 5b and c). of celiprolol. Kobayashi et al.24 reported that celiprolol might act on eNOS through the PI3K (phosphatidylinositol 30-kinase)-Akt eNOS and arginase II signaling pathway, because phosphorylation of eNOS after Both eNOS and arginase II have the same substrate. Arginase treatment with celiprolol was attenuated by wortmannin in metabolizes L-arginine to urea and ornithine, and it competes directly deoxycorticosterone acetate-salt hypertensive rats. with eNOS for L-arginine. The expression of eNOS was decreased in Sympathetic activity during sleep is known to be increased in SAS. the LV myocardium of IH mice, whereas the expression of arginase II Patients with congestive heart failure, especially those with central was increased. These changes were less marked after treatment with SAS, have a poor prognosis and show elevated plasma norepinephrine celiprolol (Figure 6). levels compared with those without SAS.25 In patients with obstructive SAS, muscle sympathetic nerve activity and plasma DISCUSSION catecholamine levels are increased, associated with systemic and/or The major features of SAS include sympathetic activation, production pulmonary hypertension.26,27 It seems that celiprolol decreases of ROS and inflammation.5,6 In the present study, celiprolol sympathetic tone and reduces BP fluctuation, which might be

Hypertension Research Celiprolol and hypoxic stress SNishiokaet al 937

Normoxia+vehicle Normoxia+celiprolol Hypoxia+vehicle Hypoxia+celiprolol

Hematoxylin- eosin stain

Sirius red stain

P<0.01 P<0.01 (µm) (%) P<0.01 P<0.01 P<0.01 P<0.05 14 1.0 12 0.8 10 0.6 8 6 0.4 diameter 4 %Fibrosis 0.2 2 Mean cardiomyocyte 0 0 (8) (8) (10) (10) (8)(8) (10) (10)

Normoxia Hypoxia Normoxia Hypoxia

Figure 3 Representative light micrographs of LV myocardium (a–h). Hematoxylin–eosin stains (original magnification Â 400, (a–d)). Sirius red stain (original magnification Â 200, (e–h)). Effect of intermittent hypoxic stress on the mean diameter of cardiomyocytes (i) and perivascular fibrosis (j)intheLV myocardium. The mean cardiomyocyte diameter and the percentage of the area of perivascular fibrosis were significantly increased in mice exposed to intermittent hypoxia, while this change was significantly suppressed by treatment with celiprolol. Values are the mean±s.e.m.

(%) P<0.01 P<0.01 Normoxia Hypoxia 14 P<0.01

Vehicle 10

4 Celiprolol

%Area of DHE fluorescence 2

0 (5) (5) (6) (5)

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Figure 4 Effect of hypoxic stress on expression of DHE in the LV myocardium. DHE staining was significantly increased in mice exposed to intermittent hypoxia, suggesting an increase in superoxide production. Treatment with celiprolol significantly decreased superoxide production in the LV myocardium. Values are the mean±s.e.m. A full color version of this figure is available at the Hypertension Research journal online.

important factors mediating its cardioprotective effect observed in the phosphorylation of nuclear factor-kB in endothelial cells, were present study. Further studies, with longer observation periods, are increased over time, which might be related to the occurrence of required to evaluate the effect of celiprolol to prevent hypertension in cardiovascular events and hypertension due to intermittent hypoxia. the future. Increased levels of TNF-a and oxidative stress promote the activation Intermittent hypoxia is known to increase the levels of of nuclear factor-kB and the development of cardiac hypertrophy. It various inﬂammatory factors. Li et al.28 reported that the serum has also been reported that intermittent hypoxic stress signiﬁcantly levels of TNF-a, interleukin-8 and interleukin-6, as well as the increases transforming growth factor-b (TGF-b), which has a major

Hypertension Research Celiprolol and hypoxic stress S Nishioka et al 938

P<0.01 P<0.01 P<0.01 P<0.01 (%) P<0.01 P<0.05 P<0.05 P<0.01 40 3.0 P<0.05 3.0 35 2.5 2.5 30 2.0 2.0 25 20 1.5 1.5

protein 15 1.0 1.0 BNP/18s ratio TNF- /18s ratio 10 0.5 0.5 5 %Area of 4-hydroxy-2-nonenal 0 0 0 (8) (8) (10) (10) (5) (5) (5) (5) (5) (5) (5) (5)

Normoxia Hypoxia Normoxia Hypoxia Normoxia Hypoxia

Figure 5 Quantitative data of immunohistochemistry and reverse transcription PCR. Intermittent hypoxia signiﬁcantly increased 4-HNE-modiﬁed protein adducts (a), and the expression of TNF-a and BNP mRNA (b, c) in the LV myocardium. Celiprolol reduced oxidative stress and suppressed the intermittent hypoxia-induced increase in myocardial levels of TNF-a and BNP mRNA. Values are the mean±s.e.m.

Normoxia+vehicle Normoxia+celiprolol Hypoxia+vehicle Hypoxia+celiprolol

Arginase II

eNOS

(%) P<0.05 P<0.01 (%) P<0.01 50 P<0.01 40 P<0.01 P<0.05 40 30 30 20 20 10 10 %Area of eNOS

%Area of Arginase II 0 0 (5) (5) (5) (5) (7) (8) (7) (9)

Normoxia Hypoxia Normoxia Hypoxia

Figure 6 Immunohistochemistry of arginase II (a–d) and eNOS (e–h) expression in the LV myocardium. Arginase II expression (i) was significantly increased and expression of eNOS (j) was significantly decreased by intermittent hypoxia, while this change was suppressed by celiprolol. Values are the mean±s.e.m. A full color version of this figure is available at the Hypertension Research journal online.

role in tissue fibrosis and inflammation. In the present study, might have a role in lessening the histological changes induced by intermittent hypoxia increased the expression of TNF-a and BNP hypoxic stress. Further investigation will be needed using other mRNA in the LV myocardium. Treatment with celiprolol significantly b-blockers (such as propranolol, atenolol and metoprolol) to suppressed TNF-a and BNP mRNA expression, and perivascular elucidate the pathophysiology of cardiomyocyte degeneration related fibrosis was also significantly reduced. We have previously reported to intermittent hypoxia-induced sympathetic activation. that expression of TGF-b mRNA in the LV myocardium was increased Recently, an angiotensin II receptor blocker (ARB) was reported to in mice treated with hypoxic stress.8 It is also reported that inhibit sympathetic nervous system activity in rats.30 It has been celiprolol decreased TGF-b in deoxycorticosterone acetate-salt shown that ARBs exhibit antioxidative and anti-inflammatory effects hypertensive rats.29 Although we did not investigate TGF-b in this in human and animals.9,31,32 We have reported that combination study, the anti-inflammatory and antioxidant effects of celiprolol therapy with celiprolol and candesartan exerts cardioprotective effects

Hypertension Research Celiprolol and hypoxic stress SNishiokaet al 939 in heart failure model rats.33 We also found that angiotensin II 14 Mehta JL, Lopez LM, Chen L, Cox OE. Alterations in nitric oxide synthase activity, expression was increased by intermittent hypoxia.10 Furthermore, superoxide anion generation, and platelet aggregation in systemic hypertension, and effects of celiprolol. Am J Cardiol 1994; 7: 901–905. olmesartan combined with nifedipine, which has antioxidative effect, 15 Miller FJ Jr, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production was effective to prevent hypoxia-induced cardiac remodeling in in vascular smooth muscle contributes to oxidative stress and impaired relaxation in 34 atherosclerosis. Circ Res 1998; 82: 1298–1305. diabetic mice. Therefore, combination therapy with celiprolol and 16 Nakano D, Hayashi T, Tazawa N, Yamashita C, Inamato S, Okuda N, Mori T, Sohmiya K, ARB (or an angiotensin-converting enzyme inhibitor) might be more Kitarura Y, Okada Y, Matsumura Y. Chronic hypoxia accelerates the progression effective in our experimental model. of atherosclerosis in apolipoprotein E-knockout mice. Hypertens Res 2005; 28: 837–845. In conclusion, the present study showed that intermittent hypoxia 17 Inamoto S, Yoshioka T, Yamashita C, Miyamura M, Mori T, Ukimura A, Matsumoto C, increased the cardiomyocyte diameter and perivascular fibrosis, at Matsumura Y, Kitaura Y, Hayashi T. Pitavastatin reduces oxidative stress and attenuates least partly, in response to an increase in oxidative stress. Celiprolol intermittent hypoxia-induced left ventricular remodeling in lean mice. Hypertens Res 2010; 33:579–586. significantly suppressed BP fluctuations, restored eNOS expression, 18 Hayashi T, Yoshioka T, Hasegawa K, Miyamura M, Mori T, Ukimura A, Matsumura Y, reduced oxidative stress and decreased superoxide production, con- Ishizaka N. Inhalation of hydrogen gas attenuates left ventricular remodeling induced by intermittent hypoxia in mice. Am J Physiol Heart Circ Physiol 2011; sequently attenuating the histological changes caused by hypoxic 301: 1062–1069. stress. These findings suggest that treatment with celiprolol might 19 Bivalacqua TJ, Hellstrom WJ, Kadowitz PJ, Champion HC. Increased expression of prevent cardiovascular events in borderline hypertensive patients with arginase II in human diabetic corpus cavernosum: in diabetic-associated erectile dysfunction. Biochem Biophys Res Commun 2001; 283:923–927. SAS. 20 Morris CR, Morris SM Jr, Hagar W, Van Warmerdam J, Claster S, Kepka-Lenhart D, Machado L, Kuypers FA, Vichinsky EP. 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